CN117230240A - InDel locus related to soybean seed oil content, molecular marker, primer and application thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular markers, and particularly relates to an InDel locus, a molecular marker, a primer and application thereof related to soybean seed oil content. The locus is located at the 48599328bp position of soybean chromosome 18, and the polymorphism is A or AGATG. The invention also discloses a molecular marker of the InDel locus, a molecular marker amplification primer, a kit containing the amplification primer and application of the kit in identifying the soybean oil component content. The invention also discloses a method for identifying the content of soybean oil, which is used for identifying the content of soybean seed oil with the accuracy of 73.22 percent. The InDel marker related to the oil content of the soybean seeds can be used for molecular marker assisted selection for soybean oil content improvement, and the application of the gene related to the oil content in fine positioning and map cloning, so that the improvement process of the excellent soybean properties is accelerated.

Description

InDel locus related to soybean seed oil content, molecular marker, primer and application thereof

Technical Field

The invention belongs to the technical field of molecular markers, and particularly relates to an InDel locus, a molecular marker, a primer and application thereof related to soybean seed oil content.

Background

Soybeans are the most common leguminous plants in the world, and belong to grain crops and important economic crops. Because the seeds are rich in high-quality oil content, the seeds have great contribution to human consumption and industrial application. From the genetic point of view, the soybean oil content-dividing character is a complex quantitative character controlled by multiple genes, and the efficiency of improving the soybean oil content-dividing content by using a conventional breeding means is low. Along with the rapid development of molecular biology technology, the gene fine regulation technology can realize the aggregation and efficient utilization of excellent alleles, is one of key technologies for breeding breakthrough large varieties, and is also a necessary means for improving the breeding capability of soybean varieties in the future. The development of excellent allele accumulation and its corresponding molecular markers is a prerequisite for achieving fine regulation and polymerization of genes.

At present, the demand for soybean is increasing, and the breeding of new varieties of high-oil soybean has become a key problem for breeders and producers. Therefore, the molecular marker of the excellent allele of the soybean oil content is discovered, so that technical reserve can be provided for the polymerization of the excellent allele of the high-oil soybean, and clear guidance can be provided for improving the soybean quality by a gene fine regulation technology.

In recent years, with the development of sequencing technology, researchers have had a more comprehensive understanding of soybean genomes. Whole Genome association analysis (Genome-WideAssociation Studies, GWAS) is an advanced method of currently studying biological genomes by typing large-scale population DNA samples for Genome-wide high-density genetic markers (such as SNPs or CNVs, etc.), thereby searching for genotypes associated with biological phenotypes. Leamy et al detected approximately 30000 Single Nucleotide Polymorphisms (SNPs) in 570 parts of wild soybean by whole genome sequencing techniques, and measured protein content, kernel oil content, and 5 fatty acid levels for these wild soybean seeds, indicating that 29 SNPs were significantly correlated with 7 compositional traits of wild soybean seeds. At present, association analysis is widely applied to plant researches such as soybean seed protein content, rice amino acid composition, triticale aluminum toxicity resistance and the like. In recent years, development of functional markers for a target trait using GWAS has become one of the hot spots in molecular biology research. The genetic improvement process of soybean high oil products can be obviously accelerated by molecular marker assisted selection.

At present, related sites influencing the oil content of soybean seeds are reported at home and abroad, most of molecular markers related to the current functional sites are derived from recombinant inbred lines or single parent-mother construction groups, and QTL (quantitative trait locus) which can be repeatedly detected in different environments or different genetic backgrounds is fewer. When these markers are applied to natural populations such as hybrid varieties and local varieties, they are often not used as molecular marker-assisted breeding, nor are the genetic contribution rates of the loci explained. An insertion/deletion (InDel) marker is a base sequence length polymorphism marker based on a PCR amplification technology, and the InDel marker is used as a molecular marker emerging In recent years and is widely applied to the fields of genetic diversity analysis, purity identification, auxiliary breeding and the like. But there are few reports on their use in whole genome association analysis.

Therefore, a means for selecting a molecular marker that is more rapid, has high genetic stability, and has good repeated results is necessary.

Disclosure of Invention

The invention aims to overcome the defect that the existing locus related to the soybean seed oil content cannot be repeatedly detected in different environments or different genetic backgrounds in the prior art, and provides InDel locus related to the soybean seed oil content, a molecular marker, a primer and application thereof.

In order to solve the technical problems, the invention adopts the following technical scheme.

The first aspect of the present invention provides an InDel site associated with soybean seed oil content, the InDel site being located at a position 48599328bp of soybean chromosome 18, the polymorphism being A or AGATG.

In a second aspect, the invention provides a molecular marker comprising the InDel site.

In a third aspect, the invention provides the molecularly-labelled amplification primer.

In some embodiments of the invention, the nucleotide sequence of the amplification primer is shown as SEQ ID NO. 3 and SEQ ID NO. 4.

In a fourth aspect, the invention provides a kit comprising said amplification primers.

In a fifth aspect, the invention provides the use of said InDel site, said molecular marker, said amplification primer or said kit for identifying soybean oil fraction.

In some embodiments of the invention, the soybean having an InDel site polymorphism a has a higher oil content than a soybean variety having an AGATG polymorphism.

In a sixth aspect, the invention provides the use of said InDel site, said molecular marker, said amplification primer or said kit for breeding high oil content soybeans.

In a seventh aspect, the present invention provides a method for identifying the content of soybean oil, said method comprising the steps of:

s1, extracting soybean genome DNA to be identified by using a CTAB method;

s2, performing PCR (polymerase chain reaction) amplification by using the soybean genome DNA as a template and using the amplification primer to obtain an amplification product;

s3, sequencing the amplified product, and judging the content of the seed oil according to the sequencing analysis result.

In some embodiments of the invention, in S3, the criterion for the determination is: when the base at 369 th position of the 5' end of the amplification product is A, judging that the soybean variety with high oil content is obtained; when the base at 369 th position of the 5' end of the amplification product is AGATG, the soybean variety with low oil content is judged.

Compared with the prior art, the invention has the following beneficial effects: the InDel marker related to the oil content of the soybean seeds can be widely applied to screening the oil content in different cultivated soybean groups and further applied to genetic improvement of soybean quality. The 48599 +/-50 kb interval of the chromosome 18 is an ideal marking interval for regulating and controlling the oil content of soybean oil, wherein the genetic contribution rate of the oil content of InDel at 48599328bp is 17.74-19.36%, and the additive effect is 0.82-0.87%. The oil content of the strain with A content of 76.27% and 73.22% at the locus in 2020 and 2022 is higher than that of the strain with AGATG content of 20.68% and 20.42%, the accuracy can reach 73.22%, the selection cost is greatly reduced, and the quality improvement efficiency is improved. The InDel marker obviously linked with the target character is screened by the whole genome resequencing technology, and the method can be used for molecular marker assisted selective breeding, so that the excellent allele polymerization process of soybean is obviously improved, and the quality improvement of the soybean seed oil content is controlled by gene fine adjustment.

Drawings

FIG. 1 shows Oil content (Oil) in the 334 relevant groups 2020 and 2022; wherein, FIG. 1A is the oil content in 334 parts of the related group of E1 (2020); FIG. 1B shows the oil content of 334 groups of E2 (2022).

FIG. 2 is a population structure diagram of the related population obtained by the admix software based on InDel.

FIG. 3 is a Manhattan plot showing the results of MLM correlation analysis of soybean oil fractions in 334 natural populations E1 (2020) and E2 (2022).

FIG. 4 is a Box chart of soybean oil fraction differences corresponding to molecular markers of soybean oil fraction in 334 natural populations for two years; fig. 4A is a Box chart of soybean oil content differences corresponding to molecular markers of soybean oil content in 334 parts of natural population in E1 (2020); fig. 4B is a Box plot of soybean oil fraction differences corresponding to molecular markers of soybean oil fraction in the E1 (2022) 334 natural population.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

In the research, 3000 parts of soybean germplasm resources which are about 40 ℃ N in China are collected and measured, and are collected and evaluated by soybean germplasm resource research team groups cultivated by soybean research institute of Jilin province agricultural sciences, and are stored in germplasm resource library of Jilin province agricultural sciences.

EXAMPLE 1 construction and trait determination of Soybean seed oil content-related populations

In this embodiment, 3000 parts of germplasm resources in a soybean germplasm resource library are used, and the source of the germplasm resources covers most of the high-latitude soybean main production areas in China, including Heilongjiang province, jilin province, liaoning province, inner Mongolia, xinjiang and the like. 3000 parts of resources are planted in the field, after the seeds are fully mature, the seeds are harvested, and 20 representative seeds are randomly selected for each variety to measure. The oil content of 3000 parts of resources accords with normal distribution in the population, and the genetic diversity index is 1.95. 334 parts of resources are extracted from the oil, the genetic diversity index of the oil content is still 1.95, and 334 resources are used as the associated population. The operation steps are as follows:

(1) Collecting 334 parts of fully mature seeds of soybean germplasm in a population, harvesting and airing the seeds (the water content is less than 15%), selecting 200-300 parts of seed samples with full grain shape, complete grains and no plant diseases and insect pests and mildew, measuring the oil content of each variety by using an Infratec-1241 grain analyzer (Danish Focus analyzer), repeating 3 times for each variety, and taking the average value as the phenotype value of the oil content of the variety.

The oil content is divided into 10 groups according to the average value (X) and standard deviation (delta) of the group oil content, wherein the class 1 is < X-2 delta, the class 10 is more than or equal to X+2delta, and the difference between the classes is 0.5 delta. The genetic diversity of each trait was evaluated by Shannon's information index (H '), H ' = - Σpilnpi, pi indicating the frequency of occurrence of the ith variation, and the genetic diversity was 1.95 by calculating the oil content of 3000 parts of resources.

33-34 parts of resources are randomly extracted from each group, 334 parts of resources are extracted in total, the genetic diversity index H 'is calculated, and when the genetic diversity index H' is equal to 1.95, the 334 resources are determined as the associated group, and the oil content of the group is normally distributed (shown in fig. 1A and 1B).

EXAMPLE 2 soybean oil fraction Whole genome correlation analysis

(1) 334 parts of resource single plant leaf DNA of the related population is extracted by a CTAB method, the DNA concentration is detected by using a Thermo nanodrop 2000, and the purity and the integrity of the DNA are detected by using 1% agarose electrophoresis.

(2) The total genome re-sequencing technology of An Nuo Youda gene limited company is utilized to carry out genome sequencing on 334 resources, and the specific operation is as follows:

the enzyme digestion scheme is as follows: enzyme digestion prediction is carried out on the published soybean reference genome by utilizing enzyme digestion prediction software, enzyme digestion is carried out on each sample genome which is qualified in detection by using endonuclease RsaI and HaeIII, and SLAF fragments with genome fragments ranging from 364 bp to 414bp are selected.

Sequencing flow: the resulting SLAF fragment was subjected to 3' -end addition treatment with Klenow fragment (3 '. Fwdarw.5 ' exo-) (NEB) and dATP at 37℃and to Dual-index sequencing adaptors, PCR amplification (PCR amplification primers shown in Table 1, wherein the upstream primer is shown in SEQ ID NO:1 and the downstream primer is shown in SEQ ID NO: 2), purification (Agencourt AMPure XP beads (Beckman Coulter, high Wycombe, UK)), mixing, cutting to select the target fragment, and sequencing with IlluminaHiSeqTM after the library quality was checked to be acceptable. To evaluate the accuracy of the library building experiments, soybean ('Williams 82': g.max wm82.a2.v1) was selected as a Control (Control) to participate in library building and sequencing.

According to the positioning result of sequencing Reads on a reference genome, the GATK performs local weight comparison (Local Realignment), GATK mutation detection, samtools mutation detection, and the steps of taking intersection mutation sites obtained by the two methods of GATK and samtools and the like so as to ensure the accuracy of InDel obtained by detection. The intersection of InDel markers obtained by the two methods is used as a final reliable InDel marker data set, and 3,306,713 groups of InDel are obtained.

TABLE 1PCR amplification primers

Primer name	Sequence(s)	Numbering device
			Upstream primer	5′-AATGATACGGCGACCACCGA-3′	SEQ ID NO:1
Downstream primer	5′-CAAGCAGAAGACGGCATACG-3′	SEQ ID NO:2

(3) Phylogenetic tree is used to represent evolutionary relationships between species, and according to the relatedness between various organisms, various organisms are arranged on a branched tree-like chart, so that the evolutionary processes and the relatedness of the organisms are represented concisely. Based on InDel, a population evolutionary tree of the sample is constructed by a MEGA5 software, neighbor-joining algorithm.

(4) The genetic structure analysis of the population can provide the source of the blood system of the individual and the composition information thereof, and is an important genetic relationship analysis tool. Based on InDel, the population structure of the samples is analyzed by an admixture software, and clustering is performed on the assumption that the number of clusters (K value) of the samples is 1-19, respectively. As shown in fig. 2, the clustering result is cross-validated, and the optimal cluster number is determined to be 13 according to the valley value of the cross-validated error rate.

(5) Based on InDel, principal component analysis (Principal components analysis, PCA) was performed by TASSEL5 software to obtain principal component clustering of the samples. Through PCA analysis, the relative approaching and the relative distant of the samples can be known, and the evolution analysis can be assisted.

(6) The correlation (relative kinshift) between two individuals in a natural population can be estimated using plink software. The genetic relationship itself is a relative value defining the genetic similarity between two specific materials and the genetic similarity between any material, and thus is defined as 0 directly when the genetic relationship value between two materials is less than 0 as a result.

(7) Based on the association population InDel molecular marker data, genetic structure data, kinship matrix data and oil content data, full genome association analysis (Genome wide association study, GWAS) is performed by using a mixed linear model ((Mixed linear model, MLM)) of GAPIT data packets, X is genotype, Y is phenotype, and finally each InDel site can obtain an association result, as shown in FIG. 3A and FIG. 3B, one point represents one InDel site, the red dotted line is the negative logarithm of 1/InDel number, the point higher than the red line indicates that the corresponding InDel marker is obviously related to the oil content, wherein the highest point on the No. 18 chromosome red line corresponds to 48599328bp InDel, and the number-log ₁₀ (p) 5.40 was used as a screening standard, and InDel marker (A/AGATG) significantly associated with oil content was obtained at 48599328bp position of chromosome 18, and detailed information is shown in Table 2.

TABLE 2 significant correlation of Oil content (Oil) of soybean seeds InDel information

Example 3 application of significant association of soybean oil fraction to InDel markers

InDel marked as 48599328bp (A/AGATG) of chromosome 18 closely linked with soybean oil content is named qOl 18-2, which is a fragment obtained by PCR amplification with qOl 18-2 primer (primer sequence is shown as SEQ ID NO:3 and SEQ ID NO:4 in Table 3) by taking genomic DNA of the material to be identified as a template; the nucleotide sequence of the qOil18-2 amplified fragment is shown in SEQ ID NO. 5 or SEQ ID NO. 6 in Table 4.

Wherein the amplification primers are shown in Table 3,

TABLE 3qOil18-2 amplification primers

Primer name	Sequence(s)	Numbering device
			qOil18-2-F	5′-CCTTAAGATGAATTAGGTCCAGATG-3′	SEQ ID NO:3
qOil18-2-R	5′-GCTTCCTTTTGCATCAGAGC-3′	SEQ ID NO:4

TABLE 4 nucleotide sequences of amplified fragments

The specific steps for auxiliary judging the oil content of the offspring of the variety by using the InDel molecular marker are as follows:

(1) Extraction of genomic DNA of a material to be identified by CTAB method

1) Fresh leaves of soybean were taken, added with liquid nitrogen and ground into powder, and a proper amount was placed into a 1.5mL centrifuge tube.

2) 0.6mL of the preheated CTAB extract was added, mixed upside down several times, mixed in a water bath at 65℃for one hour, centrifuged at 12000rpm for 15min every 15min.

3) 0.6mL 24 was added: chloroform of 1 (V/V): the isoamyl alcohol solution is inverted and mixed for 5 to 10 times and centrifuged at 10000rpm for 15min.

4) The supernatant solution was transferred to another empty centrifuge tube using 24:1 (V/V) chloroform: the isoamyl alcohol solution was re-extracted once, then 50. Mu.L of RNase (10 mg/mL) was added and left at room temperature for 30min.

5) Adding isopropanol precooled at-20deg.C, centrifuging at 5000rpm for 10min at-20deg.C in a refrigerator for 30min, and removing supernatant.

6) The mixture was washed twice with 70% ethanol. And (3) drying, dissolving with sterilized water to obtain genome template DNA, and placing the genome template DNA into a refrigerator at 4 ℃ for later use.

7) The concentration of the DNA was detected with 0.8% agarose and diluted to the working concentration for PCR amplification.

(2) And (3) performing PCR amplification by using the InDel marked primer to obtain an amplification product.

1) PCR amplification system: the total volume was 20. Mu.L, including 10-50ng of genomic template DNA 3. Mu.L, 10. Mu. L Quick Taq HSDyeMix,10pmol of primers 2. Mu.L and ddH, respectively ₂ O 3μL。

2) PCR amplification conditions: pre-denaturation at 94℃for 30s, annealing at 57℃for 30s, and extension at 72℃for 1min; cycling for 30 times; final extension at 72℃for 10min.

(3) Judging the content of the seed oil according to the sequence comparison result

Sequencing and analyzing the amplified product, wherein the average value of the oil content of the strain subgroup A at 369 th site of the 5' end of the amplified product is obviously higher than that of the strain subgroup AGATG at the site. As shown in fig. 4A and 4B, lines with a 76.27% and 73.22% a at this site had an oil content higher than 20.68% and 20.42% for the AGATG line for 2020 and 2022, with an accuracy of 73.22%, indicating that the marker was practically effective for auxiliary selection.

Example 4 application of soybean oil fraction content significant correlation InDel markers in breeding

At present, the marker is used for breeding new varieties. For example, a batch of germplasm resources which are polymerized with a plurality of high-oil and high-yield sites and have high content of own oil, such as 'dongnong 44', 'Hei 35', 'Male 04L-141', and the like, can be directly used for breeding new varieties through the markers and other oil content marker sites. In addition, when different regional excellent germplasm resources are introduced, such as 'Dongda No. 2', the molecular markers and other oil protein molecular markers are combined, so that a guiding direction can be provided for the resources to be used for local new variety breeding in future. Meanwhile, the molecular marker is used for purifying and multiplex-shaped work on the old variety, such as 'Heihe 35', and the oil content of the variety is reduced in the long-term planting process, but whether the hybrid exists or not is difficult to judge from the phenotype. Therefore, the marker and other soybean oil content molecular markers are used for joint identification to re-purify the variety, so that the oil content of the variety can restore the original properties.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (10)

1. An InDel site associated with soybean seed oil content, wherein said InDel site is located at a position 48599328bp of soybean chromosome 18, and the polymorphism is a or AGATG.

2.A molecular marker comprising the InDel site of claim 1.

3. The molecularly imprinted amplification primer of claim 2.

4. The amplification primer of claim 3, wherein the amplification primer has a nucleotide sequence set forth in SEQ ID NO. 3 and SEQ ID NO. 4.

5. A kit comprising the amplification primer of claim 4.

6. Use of the InDel site of claim 1, the molecular marker of claim 2, the amplification primer of claim 3 or the kit of claim 5 for identifying soybean oil fraction.

7. The use according to claim 6, wherein the soybean having polymorphism a at the InDel site has a higher oil content than the soybean variety having polymorphism AGATG.

8. Use of the InDel site of claim 1, the molecular marker of claim 2, the amplification primer of claim 3 or the kit of claim 5 for breeding high oil content soybeans.

9. A method for identifying the fraction of soybean oil, said method comprising the steps of:

s1, extracting soybean genome DNA to be identified by using a CTAB method;

s2, performing PCR (polymerase chain reaction) amplification by using the amplification primer of claim 3 by using the soybean genome DNA as a template to obtain an amplification product;

s3, sequencing the amplified product, and judging the content of the seed oil according to the sequencing analysis result.

10. The method of claim 9, wherein in S3, the criterion for the determination is: when the base at 369 th position of the 5' end of the amplification product is A, judging that the soybean variety with high oil content is obtained; when the base at 369 th position of the 5' end of the amplification product is AGATG, the soybean variety with low oil content is judged.